Nghiên cứu bào chế và đánh giá tác dụng tăng cường miễn dịch của cao khô lá đu đủ rừng (trevesia palmata (roxb ex lindl ) vis , họ nhân sâm araliaceae) tt tiếng anh

MINISTRY OF EDUCATION AND TRAINING MINISTRY OF DEFENCE VIETNAM MILITARY MEDICAL UNIVERSITY LÊ THỊ THANH THẢO STUDY ON THE PREPARATION PROCESS AND EVALUATION OF IMMUNE ENHANCEMENT EFFECT OF DRIED EXTRACT POWDER FROM TREVESIA PALMATA LEAVES (Roxb ex Lindl.) Vis., (Araliace ae family) Speciality : Pharmaceutical technology and Pharmaceutics Code : 9720202 SUMMARY OF PHARMACEUTICAL DOCTORAL THESIS HANOI - 2020 THIS PROJECT HAVE BEEN CONDUCTED AT VIETNAM MILITARY MEDICAL UNIVERSITY Scientific supervisor: Prof Ph.D Vo Xuan Minh Assoc Prof Ph.D Do Quyen 1st Reviewer: Prof Ph.D Nguyen Ngoc Chien 2nd Reviewer: Assoc Prof Ph.D Tran Viet Hung 3rd Reviewer: Assoc Prof Ph.D Nguyen Van Minh The thesis will be defended to the university doctoral committee in Vietnam Military Medical University at: h00, …… 2020 Possibly finding about the thesis at: National Library of Vietnam Library of Vietnam Military Medical University LIST OF SCIENTIFIC PUBLICATIONS OF THE THESIS RESULTS Le Thi Thanh Thao, Do Quyen, Nguyen Thi Ngoc, Le Thanh Son, Bui Huu Tai, Phan Van Kiem (2016), “Isolating some saponin compounds from Trevesia palmata”, Military Medicine-Pharmacy Magazine, Pharmaceutical thematic, p 23-31 Le Thi Thanh Thao, Do Quyen, Duong Binh Vu, and Phan Van Kiem (2018), “New acetylated saponins from the leaves of Trevesia palmata”, Natural Product Communications, Vol 12, N001-2, p.407-410 Le Thi Thanh Thao, Nguyen Trong Diep, Nguyen Hong Van, Nguyen Nu Huyen My, Vo Xuan Minh (2019), “Study on the extraction of total saponin from folium Trevesiae palmatae using Ultrasonic method” Military MedicinePharmacy Magazine, issue 9, 12/2019, p.175-180 Le Thi Thanh Thao, Do Quyen, Nguyen Hong Van, Vo Xuan Minh (2020), “Assessing the effect of factors and parameters of the spray drying process on the quality of dried extract powder from leaves of Trevesia palmata (Roxb ex Lindl.) Vis., Araliaceae family)” Medicine, Issue 5/2020, p 73-78 Vietnam Journal of LIST OF ABBREVIATIONS STT Abbreviation A Explanation Optical Absorbance Aer Aerosil AO Oleanolic Acid BC White blood cell C 13 Concentration Carbon-13 nuclear magnetic resonance CI Carr's compressibility index CR/DP Solid to feedstock (%) cs et al 10 11 Cyc DĐVN Cyclophosphamide Vietnamese Pharmacopoeia 12 DM/DL Solvent to material (mL/g) 13 ĐĐR Trevesia palmata 14 EtOH Ethanol 15 HMBC 16 17 HPLC HS chiết High performance liquid chromatography Extraction efficiency 18 HSPL Dilution ratio 19 HSPS Spray drying efficiency 20 HR-ESI-MS 21 22 IL INF C-NMR spectroscopy Heteronuclear multiple bond correlation spectroscopy High resolution - electrospray ionization - mass spectroscopy Interleukin Interferon 23 KLCT Body Weight 24 KTTP Particle size 25 KLRbk Apparent density 26 LD50 Lethal Dose 50 27 LOD Limit of Detection 28 29 LOQ M Limit of Quantification Mass 30 Mal Maltodextrin 31 MeOH Methanol 32 m/z Mass to charge ratio 33 OA Ovalbumin + Al(OH)3 34 35 RSD SPN Relative standard deviation Total saponin 36 SD Standard deviation 37 SE Standard error 38 STT Number 39 TCCS Institutional Standard 40 41 TCMD TD/CR Immune Enhancement Excipient to Solid ratio 42 TLC Thin layer chromatography 43 KLLTĐ The relative weight of the spleen 44 KLTƯTĐ The relative weight of the thymus 45 TB Mean 46 47 TNF UV – VIS Tumor necrosis factor Ultraviolet-visible INTRODUCTION Immunodeficiency diseases are a group of different conditions caused by one or more defects of the immune system and are clinically manifested by an increased susceptibility to infections Today, immunodeficiency diseases tend to increase with acute, recurrent or chronic consequences which are often severe Currently, the US Food and Drug Administration (US FDA) has licensed many drugs to be used clinically that have the benefit in treatment for immunocompromised patients The immunostimulants have abundant origins, which are substances derived from biological products (immunoglobulin, interferon ) by modern technology However, these synthetic pharmaceuticals are still quite expensive and often have many side effects There have been many published studies on immune enhancement effects of different plants In particular, among the group of natural compounds, saponins are the group of compounds with the most prominent immune enhancement effects The Trevesia palmata, (Rox ex Lindl.) Vis., is an evergreen medium tree, growing abundant and easily in the woodland edge or second-growth forest With the research material that are T palmata leaves, which are available in many areas in Ha Giang, Ba Vi, Cuc Phuong, Phu Tho the collection does not affect the ecological environment as well as biodiversity So far, T palmata in Vietnam has only been used based on traditional experience to take the heartwood to replace for the Tetrapanax papyrifera herb There are very few studies on this plant species in the world and no pharmaceuticals extracted from this plant with immune enhancement effects have been studied According to the above problems, the research “Study on the preparation process and evaluation of immune enhance ment effect of dried extract powder from Trevesia palmata leaves (Roxb ex Lindl.) Vis., Araliace ae family)” has been conducted with the following objectives: Formulate a process to prepare dried extract powder from T palmata leaves at a laboratory scale Establish the quality standard and initially assess the stability of dried extract powder from T palmata leaves Evaluate the immune enhancement effect and the toxicity of dried extract powder from T palmata leaves on experimental animals * The new main scie ntific contribution of the thesis: Identified the main compounds in T palmata leaves that are saponins Isolated and identified chemical structure of saponin compounds, including novel substances Developed and validated the quality standard for T palmata leaves Investigated and established the extraction procedure of T palmata leaves on a kg raw herbal material scale per batch, established the preparation process of dried extract powder of T palmata leaves using spray drying method Developed and validated the quality standard for dried extract powder from T palmata leaves The product was stable under research conditions Evaluated the immune enhancement effect on experimental animals, showing that the prepared products had abilities to improve the immune responses on experimental animals * Content and structure of the thesis: The thesis consists of 147 pages: Introduction: pages; Chapter (Overview): 27 pages; Chapter (Materials, equipment, subjects and research methods): 23 pages; Chapter (Results): 66 pages; Chapter (Discussion): 26 pages; Conclusion and Recommendation: pages * Re ference: The thesis contained 107 references, including 84 English references CHAPTER OVERVIEW The overview of the thesis has updated the following issues: - About T palmata: The characteristics and the distribution, chemical composition, biological effects and announced pharmacologica l effects, folk prescription and dose - About extract powder: the definition; classification according to Vietnam Pharmacopoeia V and European Pharmacopoeia main stages in extract powder preparation technique: extraction and drying Overview of ultrasonic extraction and spray drying, two techniques used in the thesis - Model in evaluating immune enhancement effects: in vitro and in vivo: overview about domestic and international research models CHAPTER SUBJECTS AND RESEARCH METHOD 2.1 Subjects 2.1.1 Materials T palmata leaves were collected in May 2015, washed, dried (40°C) in fan-assisted ovens to a moisture content of less than 12%, then sliced to the desired size and preserved in sealed plastic bag, placed in cool, dry storage in order to establish the institutional standard for the samples collected in Vietnam provinces Study on extracting, isolating and preparing using samples collected in Ha Giang 2.1.2 Experimental animals Adult white Swiss mice, regardless of breed, 18-22 grams weight, meeting the experimental standards provided by the National Institute of Hygiene and Epidemiology; adult white Wistar rat, regardless of breed, weight of each individual at the beginning of the experiment was 160 - 180 g, meeting the experimental standards provided by the Animal Husbandry Division of Vietnam Military Medical University 2.1.3 Reagents, chemicals, solvents Reagents, chemicals, solvents, equipment were all met the standards for research on the extraction and isolation; standard establishment; preparation; immune enhancement effect and toxicity evaluation 2.1.4 Research equipment Specific equipment: ultrasonic extraction machine (SONY MEDI SM30- CEP, Korea), PG-5 spray drying machine(China), UVVis spectrophotometer, system, high-performance hematological and liquid chromatography biochemical analyzers common reagents, chemicals, equipment and other 2.2 Re search method 2.2.1 Method of formulating the preparation process of dried extract powder from T palmata leaves 2.2.1.1 Identification of tracer Investigate chemical composition groups by specific chemical reactions T palmata leaves (5kg) were ultrasound extracted with methanol solution; fractional extraction with solvents of increasing polarization Isolate compounds by chromatographic methods Detect compounds by ultraviolet light or using reagents, checking the purity of isolated compounds by combined methods such as: TLC, HPLC, and NMR Determine the chemical structure based on physical and chemical parameters, compare with published data 2.2.1.2 Methods of setting up the quality standard of pharmaceutical raw material for T palmata leaves * Establish and validate SPN quantitative methods using UV-Vis - Principle: Total saponin in the leaves of T palmata was quantified using UV-Vis spectroscopy after the Rosenthaler’s coloring reaction SPN content was calculated according to AO - Preparation: standard solution series: AO in MeOH concentration from 100 - 300 µg/mL; test solution: 5.0 g of T palmata leaves, after chlorophyll removal, ultrasonically extract with MeOH in hours at 50°C twice, collect the extract, add the solvent to 200 mL, dilute 2.5 times; filter through 0.45µm membrane before carrying out the reaction; blank determination: MeOH - Carry out the Rosenthaler’s coloring reaction: Draw exactly 0.2 mL test solution (either standard or blank) into a test tube, then 14 Table 3.20 Results of extract, concentrate and impurity removal of 3:1 extract of T palmata leaves Index T.palmata leaves 3:1 extract 3:1 extract before after Extract removing impurity removing impurity Weight, volume 5.48 kg 170.0 L 1.83 kg 1.83 kg SPN (1) 22390 µg/g 683,98 µg/mL 156.00 ± 4.61 mg/g 205.67 ± 4.45 mg/g SPN weight 123,28 g 116,28 g 102,33 g 94.77 g 35.93% 25.24% The rate of dried residue in extract Extraction, extract concentration 83.44% efficiency based on SPN - Extraction, concentration efficiency based on SPN 77.27% SPN enrichment rate and impurity removal 131.84% (1) SPN : SPN in gram of extract solid Results showed that: SPN content in 3:1 extract before removing the impurities was 156,00 ± 4.61 mg/g After removing the impurities, concentrating and adjusting to 3:1 extract, the total saponin content was 205.67 ± 4.45 mg/g The rate of SPN enrichment was 131.84% 3.1.3.2 Prepare dried extract powder from T palmata leaves by spray drying method From the survey results, the formula and parameters of the preparation process of dried extract powder from T palmata leaves by spray drying method were selected as the followings: 15 Table 3.29 Formulation and parameters for the preparation process of dried extract powder from T palmata leaves by spray drying method No Parameter name Parameter Liquid extract for spray Liquid extract (3:1) of drying palmata leaves with excipient Spray drying excipient Aerosil Excipient to solid ratio 30% Inlet temperature 130 ± 2ºC Feed flow rate 30 mL/min CR/DP ratio 14% Atomization pressure Bar T The established process to prepare dried extract powder of T palmata leaves is described in the following diagram: 16 ĐĐR powder Meet the TCCS EtOH 50º DM/DL = 15ml/1g 1st ultrasonic extraction 700 C, 90 mins Decant, filter ĐĐR grounds DM/DL = 15ml/1g 2nd ultrasonic extraction 700 C, 90 mins EtOH collection ĐĐR extract Decant, filter ĐĐR grounds ĐĐR extract ĐĐR extracts 1+2 Concentrate, solvent recollect 3:1 ĐĐR liquid extract Settle, decant EtOH 96º Residue Fluid content1 Settle, decant Residue Fluid content Concentrate, solvent recollect 3:1 ĐĐR liquid extract Testing: - SPN: 165 - 245 mg/g - Solid proportion: 20 - 30% Figure 3.12 Scheme of the preparation stages of 3:1 liquid extract from T palmata leaves 17 3:1 ĐĐR liquid extract - Add Aer, water: Aer to solid ratio: 30%, CR/DP: 14% - Stirring homogeneously Feedstock Feed flow (30ml/mins) Spray drying Inlet temp 130 ± 20 C Atomization pressure: Bar Dried extract of ĐĐR Package Testing according to TCCS Figure 3.13 Summary cheme of preparation stage of dried extract powder from T palmata leaves by spay drying method 3.2 Re sults of setting up the quality standard and assessing the stability 3.2.1 Results of setting up the quality standard Conduct on lots of dried extract powder (each lot included batches of extract, each batch used kg of pharmaceutical raw materials); The proposed result of the institutional standard of dried T palmata extract powder include the following criteria: properties 18 (softly dried powder, pale yellowish brown, homogeneous, bitter taste, typical smell); weight loss due to drying (≤5%); total ash (15%); limits on heavy metals by AAS method (As ≤ 1.0 mg/kg; Cd ≤ 1.0 mg/kg; Hg ≤ 0.1 mg/kg; Pb ≤ 3.0 mg/kg); bacterial contamination (agar plate method according to Vietnamese Pharmacopoeia V); oleanolic acid qualification: according to TLC method; SPN content quantification ≥ 100 mg/g and ≤ 200 mg/g calculated according to AO by UV - Vis method; AO content quantification ≥ 7.5 mg/g and ≤ 12,5 mg/g by HPLC 3.2.2 Results of the evaluation of the stability of dried extract powder from T palmata leaves Results showed that all evaluation criteria after months in the accelerated aging condition and 12 months in long-term condition were all seen little changes and still reached the institutional standard of pharmaceutical 3.3 Re sults of evaluating the immune enhancement effects and the toxicity of dried extract powder from T palmata leaves on experimental animals 3.3.1 Evaluate the immune enhancement effect - On the general immunological indices: Dried extract powder from T palmata leaves at both doses of 400 and 800 mg/kg had no effect of increasing the volume of lymphoid organs, but caused increased lymphocytes T palmata extract powder at 1st dose increased the number of peripheral white blood cells and increases the number of neutrophils Whereas 2nd dose reduced the number of white blood cells and neutrophils 19 - On humoral immunity indicators: Two doses of T palmata extract powder both had the effect of increasing the concentration of IgG in peripheral blood - On the cell-mediated immunity indices: T palmata extract powder at both doses had the effect of increasing the secretion of IL2, 1st dose reduced TNF-α secretion, whereas 2nd dose caused increased secretion of TNF-α 3.3.2 Assess toxicity 3.3.2.1 Acute toxicity: Results showed that the mortality rate of mice and the level of dose of dried T palmata extract powder was strongly correlated with correlation coefficient r² = 0.9707 and the regression equation was y = 4.738e0.016x Then, LD50 = 10.54 (8.79 - 12.65) g of T palmata extract powder/kg, which was 26.35 times higher than the estimated effective dose 3.3.2.2 Semi-chronic toxicity: Monitored for 90 consecutive days, the lots of mice administered T palmata extract powder at the dose of 240 mg/kg/day and the dose of 720 mg/kg/day showed that: all mice were healthy, weight gain well and regularly; no changes in the hematological indices and the biochemical criteria for liver and kidney function evaluation; no damages on liver, spleen and kidney histopathology CHAPTER 4: DISCUSSION 4.1 About formulating preparation process 4.1.1 Identify tracer groups Identified the main group of compounds in the leaves of T palmata as saponins The thesis had isolated six triterpenoid saponin compounds; including four known saponins and two new 20 compounds; contributing to the chemistry database for this species SPNs were a group of substances with many immunological effects that have been studied Among the known compounds, kalopanaxsaponin H has been studied for a number of biological effects such as antitumor and cancer cell-selective toxicity Oleanolic acid has been proven a lot of pharmacological effects related to immunity such as anti-cancer, anti-diabetes, anti-inflammatory, liver protection, antioxidant, antibacterial, Therefore our thesis used total SPN and AO as tracers 4.1.2 Choose two quantitative methods Our thesis used two quantitative methods: SPN was quantified by UV-Vis spectroscopy method, and AO was quantified by HPLC method This is necessary because it will contribute to increased rigor and accuracy in product quality assessment The formulation and validation of both quantitative methods were carried out in accordance with ICH guidelines, showing that the quantitative process met the requirements of the analytical method 4.1.3 The institutional standard of T palmata leaves Currently, in Vietnam and in the world, there have not been many research publications on T palmata, there is no treatise on T palmata in the pharmacopeias In this study, we proposed the institutional standard based on samples collected in provinces to evaluate the quality of raw T palmata leaves 4.1.4 The process of preparing extract powder from T palmata leaves Ultrasonic extraction has been being applied more and more at laboratory to industry Comparison between extraction methods 21 shows that ultrasonic extraction method has significantly improved extraction efficiency but at lower temperature and shorter time In this study, ultrasonic extraction was used to investigate with the survey parameters: particle size of pharmaceutical raw material, type of extraction solvent, DM/DL ratio, extraction temperature and time Within the framework of the thesis, ultrasonic extraction equipment had fixed the frequency of ultrasonic waves as 60 kHz, thereby the influence of ultrasonic frequency (a factor with great influence on extraction efficiency) had not been investigated 4.1.5 Regarding extraction, concentration equipment and results of 3: extract preparation After selecting parameters of extraction procedure, extract consecutive batches, each batch of 2kg on 30-liter SM30 extraction device, 60kHz ultrasonic frequency, ultrasound heads, 400W ultrasound power Ultrasonic devices are typically characterized by ultrasonic frequencies (usually in the range of 20 - 100kHz) and ultrasonic power, but the structure of the device also affects the extraction process In this study, the ultrasonic device with the ultrasound heads is fixed to the ultrasonic frequency at 60kHz, so it is not possible to investigate the effects of ultrasonic frequencies Therefore, when scaling up equipment with other ultrasonic frequencies, it is necessary to calculate and re-evaluate the process parameters to achieve the desired extraction purpose 4.1.6 Prepare dried extract powder from T palmata leaves by spray drying In this thesis, the spray-drying method was selected to prepare dried extract powder from T palmata leaves because this is a drying technique with many advantages and currently being widely applied 22 even at the industrial scale domestically In order to develop the preparation process of dried extract powder from T palmata leaves by spray-drying method, it is necessary to examine the parameters of the device (inlet gas temperature, feed flow rate) and the parameters spraying feed (solid ratio, type, excipient ratio) The parameters of the product to be assessed were: weight loss due to drying, hygroscopic property, apparent density, flowability, spray drying efficiency, active ingredient content, and efficiency - The excipients selected for the survey were Mal, Aer, a mixture of Mal and Aer, Starch 1500, lactose Aer was chosen because it has the effect of protecting active ingredients, regulating the flowability, improving the moisture absorption - Results of the investigations showed that when the Aer ratio increased, both the spray drying efficiency and other physicochemical indicators of the product were improved, the collection efficiency of the active ingredient also increased slightly, proving that Aer showed certain protective ability for active ingredients, but the total saponin content tended to decrease, this is due to the concentration dilution phenomena In this study, selecting an excipient ratio of 30% was appropriate - Investigation results showed that spray drying formulas at different temperatures and feed flow rates had certain influences on the efficiency, physical and chemical properties of the product Excessive drying temperature was also able to affect the stability of active ingredients, the investigation results showed that the content and collection efficiency of active ingredient tended to decrease when the temperature increased to 150°C - The obtained extract of T palmata leaves was concentrated to 3: extract powder with solid ratio of about 25%, but after spray drying this ratio was adjusted to 10 - 16% Because if the solid ratio 23 is too high, it will increase the viscosity of the spraying fluid, therefor it will be difficult to make droplets during spray drying, reducing spray drying efficiency Results showed that when increasing the solid to feedstock ratio, almost no effect on the SPN content and collection efficiency was seen, but humidity, hygroscopic property, flowability tended to increase In particular, the solid ratio of 14 - 16% was the most appropriate 4.2 About setting up the quality standard and assessing stability 4.2.1 Setting up institutional standard After researching on preparation of dried extract powder from T palmata leaves, the thesis had set a institutional standard to standardize product quality The lower limit criterion of quantification, we used approximately 70% of the actual quantitative investigation value … 4.2.2 Assessing stability Results of stability assessment after months in accelerated aging condition and 12 months in long-term condition, did not notice any significant change in physical and chemical stability Only in the powder form, after a long time of storage, the powder tended to coalesce, but when gently shaking the bottle or stirring with a glass rod, the powder broke down and released the initial particles 4.3 About immune enhance ment effect and toxicity 4.3.1 Immune enhancement effect Basis of choosing dose: Quantitative results showed that the SPN content in dried extract powder of T palmata leaves was 150.16 ± 3.79 mg/g; The SPN content in the extract by water decoction method was 9,98 ± 0,59 mg/g, so the dried extract powder/medicinal ratio was about 15/1 According to Vo Van Chi, the recommended 24 medicinal dose was 20 - 30 g of T palmata leaves/day (25 g used by the decoction method corresponding to 1.66 g of dried/day (≈ 1700 mg)) Therefore the expected human dose is 1700 mg/person/day The average weight of a person is 50 kg, then the expected dose per for 50 kg person will be 34 mg/kg/day According to WHO guidelines on the extrapolate dose among species, the conversion factor of equivalent dose in mice is 12, then the expected effective dose in mice is 400 mg/kg/day Similarly, the conversion factor of equivalent dose in mice is 07, then the expected effective dose in rats is 240 mg/kg/day Immune enhancement effect + T.palmata at a dose of 400 mg/kg (expected clinical dose) showed immune-boosting effect quite clearly since it tended to increase the number of peripheral total white blood cells, neutrophils, lymphocytes, IgG concentration, and also increased mouse feet thickness in patch test to OA antigen, increased IL-2 concentration, decreased TNF-α + At high dose (800 mg/kg, times higher than the expected dose), T palmata showed unclear side effects while increasing the number of lymphocytes, IgG concentration, mouse feet thickness in patch test to OA antigen, and IL-2 level However, it reduced the total number of white blood cells, neutrophils and increased TNF-α level This can be explained that high doses can cause damage to certain tissues and organs, causing white blood cell decrease This is a common feature of immune-boosting drugs as the immune response mechanism is extremely complicated In the future, if using 25 pharmaceuticals from T palmata as medications or health products, dose control should be particular attention 4.3.2 Toxicity of Trevesia palmata dried extract powder According to the guidelines of the World Health Organization and the regulations of the Ministry of Health of Vietnam for drugs of botanical origin, it must be evaluated for acute and semi-chronic toxicity on animals before being used in human being Trevesia palmata dried extract is a preparation made from medicinal herbs, with the development orientation for human using, so the toxicity study including acute and semi-chronic toxicity on experimental animals is obligatory According to US regulations, if the intended duration of human use is more than 30 days, the study period for semi-chronic toxicity on animals is months Thus, conducting a semi-chronic toxicity for a 3-month period to ensure the safety of the products to be used on human for more than 30 days on a daily basis 26 CONCLUSION About the preparation proce ss of dried extract powder of T palmata leaves - Identified the main group of substances in the leaves of T palmata as triterpenoid saponins From kg of T palmata leaves, the chemical structure of six saponin compounds were isolated and identified, including two new compounds named acetyltrevesiasaponin B (11.0 mg) and acetyltrevesiasaponin A (15.0 mg); four known compounds: Ilekudinosid C (100.3mg), kalopanaxsaponin H (90.0 mg), davisianosid B (140.0 mg) and oleanolic acid (167.0 mg) - Developed and validated the institutional standard of pharmaceutical raw material for T palmata leaves, including indicators: AO qualification by TLC method, total saponin quantification by UV - Vis (≥ 15 mg/g) and AO quantification by HPLC (≥ 0.75 mg/g) - Formulated the process of extracting extract powder from T palmata leaves with the scale of kg of pharmaceutical raw material/batch with the following conditions: powder size of T palmata leaves 0.5 - mm; ultrasonic extraction, solvent EtOH 50º, temperature 70°C, two times of extraction, DM/DL ratio 15mL/1g for two times, extraction period 90 minutes/time Then concentrate extract powder at 70 ± 5°C and 60 - 65 mmHg - Formulate the preparation process of dried extract powder of T palmata leaves with the following parameters: spray-drying fluid: (3:1) extract powder of T palmata leaves; spray drying excipients: 27 Aerosil; excipient to solid ratio: 30%; inlet temperature: 130 ± 2ºC; feed flow rate: 30 mL/minute; solid rate in spray solution: 14%; atomization pressure: Bar About the baseline standard and the stability of dried extract powder T palmata leaves - Developed and validated the institutional standard for dried extract powder from T palmata leaves, including criteria: AO qualification by TLC method; total saponin quantification by UV Vis (≥ 100 mg/g and ≤ 200 mg/g) and AO quantification by HPLC (≥ 7.5 mg/g and ≤ 12,5 mg/g) - Investigated preliminary the stability of dried extract powder of T palmata leaves for months in accelerated aging condition and 12 months in long-term condition All preparations were stable under research conditions About the immune enhancement effect and the toxicity of dried extract powder of T palmata leave s - Evaluated the immune enhancement effect of dried extract powder of T palmata leaves at two doses of 400 and 800 mg/kg on white mice At the dose of 400 mg/kg, T palmata extract powder showed immune enhancement effect more clearly than at 800 mg dose + On the general immunological indices: Dried extract powder of T palmata leaves in both doses did not cause to increase the volume of lymphoid organs and increase lymphocytes T palmata extract powder at the dose of 400 mg/kg increased the number of peripheral white blood cell and the number of neutrophils Whereas 28 at the dose of 800 mg/kg, T palmata extract powder caused decreases in the number of total white blood cells, and neutrophils + On humoral immunity indicators: Two doses of T palmata extract powder both had the effect of increasing the concentration of IgG in the peripheral blood + On the cell-mediated immunity indices: T palmata extract powder at two doses both had made an increase in the secretion of IL-2, the dose of 400 mg/kg had the effect of reducing TNF-α secretion, while 800 mg/kg dose caused an increase in TNF-α secretion - Assessed the toxicity of dried extract powder from T palmata leaves: + Acute toxicity: LD50 of oral T palmata extract powder was 10.54 (8.79 - 12.65) g/kg of white mouse weight, 26.35 times higher than the expected effective dose + Semi-longterm toxicity: T palmata extract powder was safe at doses of 240 and 720 mg/kg of white rat weight per day for 90 consecutive days RECOMMENDATION Investigating T palmata leaves at many other locations to develop the treatise on the quality standard of T palmata leaves in Vietnam Pharmacopoeia Investigating the content of compounds isolated from the leaves of T palmata and evaluating the immune enhancement effect of these compounds Developing immune-boosting products from dried extract powder of T palmata leaves ... dried extract powder from leaves of Trevesia palmata (Roxb ex Lindl. ) Vis. , Araliaceae family)” Medicine, Issue 5/202 0, p 73-78 Vietnam Journal of LIST OF ABBREVIATIONS STT Abbreviation A Explanation... the solvent (70ºC, reduced pressure, concentrate to 3:1 extract, settle for 24h, decant) The residue was added with EtOH times, settle, decant, filter adjustably to 3:1 extract) The results obtained... Thi Thanh Thao, Do Quyen, Nguyen Thi Ngoc, Le Thanh Son, Bui Huu Tai, Phan Van Kiem (201 6 ), “Isolating some saponin compounds from Trevesia palmata? ? ?, Military Medicine-Pharmacy Magazine, Pharmaceutical